The objective of this project is to understand with greater clarity the role of synaptic vesicles and calcium ion in the release of neurotransmitter. Colloidal gold will be used as a label to trace the fate of synaptic vesicle membrane in nerve terminals tetanically stimulated at 10 Hz for 5 and 20 minutes at room temperature and at 10 degrees C using OsO4 as an electron microscopic fixative. Split cutaneous pectoris muscles of the frog will be used so that the same junctions which have been monitored electrophysiologically can be examined for changes in their fine structure. Colloidal gold with different charges and surface properties will also be used to trace the fate of synaptic vesicle membrane under these same conditions. These studies are aimed at resolving significant ambiguities that now exist on the nature of synaptic vesicle recycling and furthering the understanding of this important mechanism. Elevated temperature (35 degrees C) will be used as a means of examining the relation between the concentration of extracellular calcium and ability of the frog neuromuscular junction to recycle its synaptic vesicles during the spontaneous release of transmitter. Elevated temperature will also be used to develop a method of determining the number of synaptic vesicles in a nerve terminal without damage to the terminal.